Functional Delineation of a Protein–Membrane Interaction Hotspot Site on the HIV-1 Neutralizing Antibody 10E8
Antibody engagement with the membrane-proximal external region (MPER) of the envelope glycoprotein (Env) of HIV-1 constitutes a distinctive molecular recognition phenomenon, the full appreciation of which is crucial for understanding the mechanisms that underlie the broad neutralization of the virus. Recognition of the HIV-1 Env antigen seems to depend on two specific features developed by antibodies with MPER specificity: (i) a large cavity at the antigen-binding site that holds the epitope amphipathic helix; and (ii) a membrane-accommodating Fab surface that engages with viral phospholipids. Thus, besides the main Fab-peptide interaction, molecular recognition of MPER depends on semi-specific (electrostatic and hydrophobic) interactions with membranes and, reportedly, on specific binding to the phospholipid head groups. Here, based on available cryo-EM structures of Fab-Env complexes of the anti-MPER antibody 10E8, we sought to delineate the functional antibody-membrane interface using as the defining criterion the neutralization potency and binding affinity improvements induced by Arg substitutions. This rational, Arg-based mutagenesis strategy revealed the position-dependent contribution of electrostatic interactions upon inclusion of Arg-s at the CDR1, CDR2 or FR3 of the Fab light chain. Moreover, the contribution of the most effective Arg-s increased the potency enhancement induced by inclusion of a hydrophobic-at-interface Phe at position 100c of the heavy chain CDR3. In combination, the potency and affinity improvements by Arg residues delineated a protein-membrane interaction site, whose surface and position support a possible mechanism of action for 10E8-induced neutralization. Functional delineation of membrane-interacting patches could open new lines of research to optimize antibodies of therapeutic interest that target integral membrane epitopes.
- Instituto Biofisika (CSIC-UPV/EHU), University of the Basque Country (UPV/EHU), 48080 Bilbao, Spain.
- Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), 48080 Bilbao, Spain.
- Department of Physiology, Faculty of Pharmacy, University of the Basque Country (UPV/EHU), Paseo de la Universidad, 7, 01006 Vitoria-Gasteiz, Spain.
- Laboratory of Global Healthcare, School of Pharmaceutical Sciences, Kyushu University, Fukuoka 819-0395, Japan.
- Leibniz Institute of Photonic Technology e.V., 07745 Jena, Germany.
- Faculty of Physics and Astronomy, Institute of Applied Optics and Biophysics, Friedrich Schiller University Jena, 07743 Jena, Germany.
- Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX1 2JD, UK.
- Ikerbasque, Basque Foundation for Science, 48013 Bilbao, Spain.
- Pharmacokinetic, Nanotechnology and Gene Therapy Group, Faculty of Pharmacy, University of the Basque Country UPV/EHU, 01006 Vitoria-Gasteiz, Spain.
- Microbiology, Infectious Disease, Antimicrobial Agents, and Gene Therapy, Bioaraba, 01006 Vitoria-Gasteiz, Spain.